Abstract The epithelia of the intestine and colon provide a barrier against gut microbiota, which can modify the growth, metabolism, and cell fate decisions of epithelial cells in profound ways. Emerging evidence suggests that, to meet this unique challenge, the epithelial cells are quite plastic, capable of dedifferentiating into stem cells in order to maintain the tissue?s regenerative capacities and proper homeostasis. However, the mechanisms by which (1) epithelial cells respond to different microbes and their derived metabolites and (2) the epigenetic regulatory elements that drive this plasticity are largely unknown. Addressing these two fundamental questions will enable us to understand the robustness of epithelial stem cell regulation. Within the scope of the R35, we are developing platform technologies to address these two fundamental questions. First, we have been developing a 2D organoid based screening assay to investigate host- microbial interactions in a high-throughput fashion. The 2D fluorescent organoid assay allows us to plate different gut bacterial strains on the apical (luminal) side of monolayer organoid cells, while fluorescent probes enable readout of bacterial and host cell states during their interactions. Second, we have been developing an epigenetic CRISPR-dCAS9 organoid screening platform. Guided by epigenetic profiling including ATAC-seq and Mint-ChIP, high-throughput guide RNA (gRNA) libraries are designed to target lineage-specific chromatin regions. By measuring enriched gRNAs from regenerated stem cells isolated based on fluorescent markers, we will systematically identify regulatory elements that drive de-differentiation and plasticity. To accomplish these two tasks, we are requesting two essential pieces of equipment. First, we need a fluorescent plate reader, which will allow us to measure the fluorescent signals from reporters in bacteria, organoid cells, and the CRISPR/dCAS9 constructs. We were using the plate reader in the departmental teaching lab previously, which has recently moved and is now unavailable. Second, we hope to purchase an IncuCyte imaging system, which provides automated capabilities for time-series quantification of bacterial/organoid growth and lineage commitment dynamics. The equipment will enable us to develop the two organoid-based screening platforms and to identify novel mechanisms that regulate host-microbial interactions and cell fate plasticity.